Amyloid fibrils nucleated and organized by DNA origami constructions.

Amyloid fibrils are ordered, insoluble protein aggregates that are associated with neurodegenerative conditions such as Alzheimer's disease. The fibrils have a common rod-like core structure, formed from an elongated stack of ß-strands, and have a rigidity similar to that of silk (Young's modulus of 0.2-14 GPa). They also exhibit high thermal and chemical stability and can be assembled in vitro from short synthetic non-disease-related peptides. As a result, they are of significant interest in the development of self-assembled materials for bionanotechnology applications. Synthetic DNA molecules have previously been used to form intricate structures and organize other materials such as metal nanoparticles and could in principle be used to nucleate and organize amyloid fibrils. Here, we show that DNA origami nanotubes can sheathe amyloid fibrils formed within them. The fibrils are built by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin and a DNA origami construct is used to form 20-helix DNA nanotubes with sufficient space for the fibrils inside. Once formed, the fibril-filled nanotubes can be organized onto predefined two-dimensional platforms via DNA-DNA hybridization interactions.

Strategy for naturelike designer transcription factors with reduced toxicity.

For clinical applications, the biological functions of DNA-binding proteins require that they interact with their target binding site with high affinity and specificity. Advances in randomized production and target-oriented selection of engineered artificial DNA-binding domains incited a rapidly expanding field of designer transcription factors (TFs). Engineered transcription factors are used in zinc-finger nuclease (ZFN) technology that allows targeted genome editing. Zinc-finger-binding domains fabricated by modular assembly display an unexpectedly high failure rate having either a lack of activity as ZFNs in human cells or activity at "off-target” binding sites on the human genome causing cell death. To address these shortcomings, we created new binding domains using a targeted modification strategy. We produced two SP1 mutants by exchanging amino acid residues in the alpha-helical region of the transcription factor SP1. We identified their best target binding sites and searched the NCBI HuRef genome for matches of the nine-base-pair consensus binding site of SP1 and the best binding sites of its mutants. Our research concludes that we can alter the binding preference of existing zinc-finger domains without altering its biological functionalities.

HolT Hunter: software for identifying and characterizing low-strain DNA Holliday Triangles.

Synthetic DNA nanostructures are most commonly held together via Holliday junctions. These junctions allow for a wide variety of different angles between the double helices they connect. Nevertheless, only constructs with a very limited selection of angles have been built, to date, because of the computational complexity of identifying structures that fit together with low strain at odd angles. I have developed an algorithm that finds over 95% of the possible solutions by breaking the problem down into two portions. First, there is a problem of how smooth rods can form triangles by lying across one another. This problem is easily handled by numerical computation. Second, there is the question of how distorted DNA double helices would need to be to fit onto the rod structure. This strain is calculated directly. The algorithm has been implemented in a Mathematica 8 notebook called Holliday Triangle Hunter. A large database of solutions has been identified. Additional interface software is available to facilitate drawing and viewing models.

A zwitterion-DNA coating stabilizes nanoparticles against Mg2+ driven aggregation enabling attachment to DNA nanoassemblies.

Plasmonics and photonics demand new methods for the controlled construction of nanoparticle (NP) arrays. Complex, low-symmetry configurations of DNA-functionalized NPs are obtained by connection to scaffolds of branched and folded DNA nanostructures. However, the stabilization of these branched structures by Mg(2+) counterions also drives the uncontrolled aggregation of NPs. We demonstrate, using a two-dimensional DNA scaffold, that derivatizing gold nanoparticles (AuNPs) with zwitterionic ligands overcomes this problem.

Switching binary states of nanoparticle superlattices and dimer clusters by DNA strands.

Nanoscale components can be self-assembled into static three-dimensional structures, arrays and clusters using biomolecular motifs. The structural plasticity of biomolecules and the reversibility of their interactions can also be used to make nanostructures that are dynamic, reconfigurable and responsive. DNA has emerged as an ideal biomolecular motif for making such nanostructures, partly because its versatile morphology permits in situ conformational changes using molecular stimuli. This has allowed DNA nanostructures to exhibit reconfigurable topologies and mechanical movement. Recently, researchers have begun to translate this approach to nanoparticle interfaces, where, for example, the distances between nanoparticles can be modulated, resulting in a distance-dependent plasmonic response. Here, we report the assembly of nanoparticles into three-dimensional superlattices and dimer clusters, using a reconfigurable DNA device that acts as an interparticle linkage. The interparticle distances in the superlattices and clusters can be modified, while preserving structural integrity, by adding molecular stimuli (simple DNA strands) after the self-assembly processes has been completed. Both systems were found to switch between two distinct rigid states, but a transition to a flexible device configuration within a superlattice showed a significant hysteresis.

Metallic nanoparticles used to estimate the structural integrity of DNA motifs.

Branched DNA motifs can be designed to assume a variety of shapes and structures. These structures can be characterized by numerous solution techniques; the structures also can be inferred from atomic force microscopy of two-dimensional periodic arrays that the motifs form via cohesive interactions. Examples of these motifs are the DNA parallelogram, the bulged-junction DNA triangle, and the three-dimensional-double crossover (3D-DX) DNA triangle. The ability of these motifs to withstand stresses without changing geometrical structure is clearly of interest if the motif is to be used in nanomechanical devices or to organize other large chemical species. Metallic nanoparticles can be attached to DNA motifs, and the arrangement of these particles can be established by transmission electron microscopy. We have attached 5 nm or 10 nm gold nanoparticles to every vertex of DNA parallelograms, to two or three vertices of 3D-DX DNA triangle motifs, and to every vertex of bulged-junction DNA triangles. We demonstrate by transmission electron microscopy that the DNA parallelogram motif and the bulged-junction DNA triangle are deformed by the presence of the gold nanoparticles, whereas the structure of the 3D-DX DNA triangle motif appears to be minimally distorted. This method provides a way to estimate the robustness and potential utility of the many new DNA motifs that are becoming available.

Assembly pathway analysis of DNA nanostructures and the construction of parallel motifs.

We present a system for analyzing the assembly pathway of DNA nanostructures. This enables the identification, explanation, and avoidance of obstacles to proper structure formation. Potential problems include strand end-pinning and misfolding caused by the structural bias of nominally flexible junctions. We have used this system to guide the construction of parallel motifs that had previously, for unknown reasons, resisted assembly.

Double cohesion in structural DNA nanotechnology.

Double cohesion has proved to be a useful tool to assemble robust 2D arrays of large tiles. Here we present a variety of examples showing the utility of this approach. We apply this principle to the 3 types of 2D lattice sections of arrays whose individual tiles are inherently 3 dimensional, because they contain three vectors that span 3-space. This application includes motifs which are based on the tensegrity triangle, the six-helix bundle motif and on three skewed triple crossover molecules. All of these designs have the potential to form 3 dimensional structures if all three directions of propagation are allowed. If one direction is blunted, 2D arrays form, and all 3 combinations are presented here. In addition, a large parallelogram array that was not attainable previously using single duplex cohesion was also constructed using double cohesion. For comparison, arrays which use another type of double cohesion, double paranemic (PX) cohesion are also presented. Double cohesion of sticky ends proved to be the more effective tool to assemble large motifs into arrays.

Design of minimally strained nucleic Acid nanotubes.

A practical theoretical framework is presented for designing and classifying minimally strained nucleic acid nanotubes. The structures are based on the double crossover motif where each double-helical domain is connected to each of its neighbors via two or more Holliday-junction-like reciprocal exchanges, such that each domain is parallel to the main tube axis. Modeling is based on a five-parameter characterization of the segmented double-helical structure. Once the constraint equations have been derived, the primary design problem for a minimally strained N-domain structure is reduced to solving three simultaneous equations in 2N+2 variables. Symmetry analysis and tube merging then allow for the design of a wide variety of tubes, which can be tailored to satisfy requirements such as specific inner and outer radii, or multiple lobed structures. The general form of the equations allows similar techniques to be applied to various nucleic acid helices: B-DNA, A-DNA, RNA, DNA-PNA, or others. Possible applications for such tubes include nanoscale scaffolding as well as custom-shaped enclosures for other nano-objects.

Architecture with GIDEON, a program for design in structural DNA nanotechnology.

We present geometry based design strategies for DNA nanostructures. The strategies have been implemented with GIDEON-a graphical integrated development environment for oligonucleotides. GIDEON has a highly flexible graphical user interface that facilitates the development of simple yet precise models, and the evaluation of strains therein. Models are built on a simple model of undistorted B-DNA double-helical domains. Simple point and click manipulations of the model allow the minimization of strain in the phosphate-backbone linkages between these domains and the identification of any steric clashes that might occur as a result. Detailed analysis of 3D triangles yields clear predictions of the strains associated with triangles of different sizes. We have carried out experiments that confirm that 3D triangles form well only when their geometrical strain is less than 4% deviation from the estimated relaxed structure. Thus geometry-based techniques alone, without detailed energetic considerations, can be used to explain certain general trends in DNA structure formation. We have used GIDEON to build detailed models of double crossover and triple crossover molecules, evaluating the non-planarity associated with base tilt and junction misalignments. Computer modeling using a graphical user interface overcomes the limited precision of physical models for larger systems, and the limited interaction rate associated with earlier, command-line driven software.

Experiments in Structural DNA Nanotechnology: Arrays and Devices.

In recent years, the chemistry of DNA has expanded from biological systems to nanotechnology. The generalization of the biological processes of reciprocal exchange leads to stable branched motifs that can be used for the construction of DNA-based geometrical and topological objects, arrays and nanomechanical devices. The information in DNA is the basis of life, but it can also be used to control the physical states of a variety of systems, leading ultimately to nanorobotics; these devices include shape-changing, walking and translating machines. We expect ultimately to be able to use the dynamic information-based architectural properties of nucleic acids to be the basis for advanced materials with applications from nanoelectronics to biomedical devices on the nanometer scale.